Ampkinone

Kinase experiment:

Total AMPK activity is measured using a synthetic SAMS peptide substrate and [γ-32P]ATP. Briefly, 500 μg of protein extract is incubated with anti-AMPK-α1 and α2 antibodies for 2 h at 4°C. Protein A/G sepharose and agarose are added, and the mixtures are incubated for 3 h at 4°C. After the samples are washed three times with RIPA buffer, the activity is assessed in AMPK reaction buffer containing 20 mM HEPES-NaOH (pH 7.0), 0.4 mM Dithiothreitol, and 0.01% Brij-35, with or without 300 μM AMP. The immune complexes are added to 23 μL of reaction buffer per assay, and then 7 μL of SAMS substrate peptide (HMRSAMSGLHLVKRR, 100 μM final concentration) and 10 μL of ATP mixture (an aliquot of 1 μCi/μL: 90 μL of 75 mM magnesium chloride, 500 μM unlabeled ATP in 20 mM MOPS, pH7.2, 25 mMβ-glycerophosphate, 5 mM EGTA, 1 mM sodium orthovanadate, and 1 mM Dithiothreitol) are added, and the mixture is incubated at 30°C for 15 min. After 35 μL is spotted onto the center of P81 paper, the paper is washed three times with 0.75% phosphoric acid and once with acetone for 5 min. The samples are read using a scintillation counter[1].

Animal experiment:

Mice[1]C57BL/6J mice are used and housed individually in a room maintained at 25°C on a 12/12 h light/dark schedule. For dietinduced obesity (DIO) mice, 4-week-old male C57BL/6J mice are fed a high-fat diet (HFD, 60% calories from fat) ad libitum for 8 weeks. Ampkinone in polyethylene glycol (PEG400) or vehicle is administered subcutaneously at 10 mg/kg body weight per day for 1 month. Body weight and food intake are measured every 3 days. All animals are given an insulin tolerance test (ITT) and sacrificed at 30 days. Liver weight and fat masses are measured, sectioned, and stained with Oil-red O and H&E.

References:

[1]. Oh S, et al. Antidiabetic and antiobesity effects of Ampkinone (6f), a novel small molecule activator of AMP-activated protein kinase. J Med Chem. 2010 Oct 28;53(20):7405-13.